Engineering Marchantia polymorpha chloroplasts for the production of high-value specialized terpenes

Through this project, we aim to pioneer metabolic engineering in chloroplasts of M. polymorpha, and assess the potential of exploiting them as a production chassis for high-value specialized terpenes. As a proof-of-concept the heterologous production of geraniol, amorpha-4,11-diene and β-amyrin in chloroplasts will be evaluated.

The Idea

Land plants produce a huge variety of chemicals. Some of these are primary metabolites essential for basic metabolic processes involved in plant growth, development and reproduction. The other non-essential chemicals are called secondary metabolites, specialized metabolites or natural products, and have important ecological functions primarily in plant defence against herbivores, pests and pathogens. These natural products are not only useful to the plants that produce them, but also have powerful physiological effects in humans [1]. Plant parts and extracts have been traditionally used in different cultures for the treatment of a wide range of ailments. In modern medicine, plant natural products, their derivatives and analogues represent more than half of all clinically used therapeutics. Plant natural products are also consumed by humans in everyday foods as flavours and fragrances. This makes land plants a very rich source of natural products with diverse and useful properties for humankind. However, the use of many natural products is restricted by their limited accumulation in the plant, slow growth rate of the plant and varying levels of compound accumulation that are susceptible to geographical and environmental conditions. In addition, extraction of natural products most often involves the use of destructive methods on the source and can be uneconomical. Hence, engineering heterologous hosts that are easy to manipulate, maintain and cultivate for the production of natural products with economic value is of great interest [2].

The terpenes represent a large class of structurally diverse plant natural products. They are derived from the repetitive fusion of isoprene (C5) units, and are classified as hemi- (C5), mono- (C10), sesqui- (C15), di- (C20), tri- (C30), tetra- (C40), or poly- (C>40) terpenes based on the number of units they contain. Because of their structural variety, the terpenes display a wide spectrum of biological activities [3]. For instance, the monoterpene geraniol, the sesquiterpene amorpha-4,11-diene and the triterpene β-amyrin are precursors of the potent anti-cancer drug vincristine, the anti-malarial drug artemisinin, and bioactive triterpenoid saponins, respectively. In plant cells terpene biosynthesis is highly compartmentalized [2]. The precursors for hemi-, mono-, di-, tetra- and polyterpene synthesis are generated in the chloroplast, while the sesqui- and triterpene precursors are synthesized in the cytoplasm. Ongoing research in the Osbourn lab (JIC) supports the notion that triterpene production in Nicotiana benthamiana can be improved by directing its biosynthesis to non-native cellular compartments, like the chloroplast.

The thalloid liverwort Marchantia polymorpha has recently received marked attention as a basal multicellular plant chassis. On account of its simple propagation, high regenerative capacity, and established tools for nuclear and chloroplast transformation, M. polymorpha shows great promise as a heterologous host for metabolic engineering. Liverworts naturally synthesize enzymes and secondary metabolites of commercial relevance, including antifungal [4], antimicrobial [5] and anticancer [6] agents. In addition, suspension cultures of liverworts have been used in the biotransformation of organic substrates and photoautotrophic growth conditions have been established allowing their large-scale cultivation in bioreactors [7]. In M. polymorpha, although the production of specialized terpenes has not been reported, several isoprenoid biosynthesis enzymes catalyzing the synthesis of terpene precursors have been identified and shown to have similar subcellular compartmentalization as land plants [8]. Through this project, we aim to pioneer metabolic engineering in chloroplasts of M. polymorpha, and assess the potential of exploiting them as a production chassis for high-value specialized terpenes. As a proof-of-concept the heterologous production of geraniol, amorpha-4,11-diene and β-amyrin in chloroplasts will be evaluated.

Project Outputs

Summary of the project's achievements and future plans

Original proposal and application

Engineering Marchantia polymorpha chloroplasts for the production of high-value specialized terpenes

Summary

Originally, three independent operon-like synthetic constructs should be built to achieve de novo synthesis of mono-, sesqui- and triterpenes in M. polymorpha chloroplasts. GoldenGate modules of coding sequences to be expressed in M. polymorpha were synthesized. However, two major issues were encountered during the project, including problems with transforming M. polymorpha chloroplasts with large constructs, and an assembly defect of the 2A peptide system used for generating the clusters. To circumvent these obstacles, constructs allowing nuclear transformation of M. polymorpha and subsequent chloroplast targeting of the proteins were designed and a new 2A peptide system has been created and is currently being evaluated.

Report and Outcomes

Originally, three independent operon-like synthetic constructs were designed for the de novo synthesis of mono-, sesqui- and triterpenes in M. polymorpha chloroplasts (Genbank files pChloro). The coding sequence of each heterologous gene to be expressed in M. polymorpha was subjected to the Marchantia chloroplastic codon optimization tool previously available on the Integrated DNA Technologies (IDT) website (https://eu.idtdna.com/CodonOpt, “Chloroplast Marchantia polymorpha (liverwort)” tool). The codon-optimized sequences were used to design and synthesize Golden Gate modules (Genbank files EC81672 to EC81687, EC81704 and EC81705) compatible with the 2A peptide building blocks available at the JIC at the start of the project. From an early stage in the project it became evident that Marchantia chloroplast transformation using such large constructs could be an issue. As our objective was to synthesize terpenes in chloroplasts, three additional constructs allowing nuclear transformation of M. polymorpha and subsequent chloroplast targeting of the proteins were designed (Genbank files pNucl). For chloroplast targeting we used the M. polymorpha chloroplast-transit peptide, previously characterised at the University of Cambridge (module EC81783). A set of six constructs was built to test the ability of the designed vectors to produce mono-, sesqui- and triterpenes in Nicotiana benthamiana agroinfiltration-based transient expression assay, prior to stable transformation of M. polymorpha. The construction of these vectors revealed a major flaw in the 2A peptide system available at that time in JIC. The 4bp linkers that define the ordered assembly of the 2A-flanked sequences into the Golden Gate binary expression vectors were very similar to each other (only one base pair difference between each linker), which resulted in the creation of random incorrect assemblies. To overcome this, a new set of 2A peptide modules and terpene biosynthetic modules, fully compatible with the plant synthetic biology common syntax (Patron et al., 2015) was created (EC81823 to EC81828, EC81831, EC81832 and EC81846 to EC81855). The efficiency of the 2A modules to mediate production of independent fluorescent proteins was successfully assessed using an agrobacterium-mediated N. benthamiana transient expression assay (Figure 1, construct EC80046). Also the assembly of the six large operon-like constructs using the newly created modules was efficiently achieved (Genbank files EC81833 to EC81835 and EC81866 to EC81868). Nevertheless, none of these six constructs were functional when transiently expressed in N. benthamiana. Confocal microscopy analysis of the infiltrated leaves revealed the absence of accumulation of mTurquoise fluorescent reporter present at the end of each construct. Furthermore, Gas Chromatography - Mass Spectrometry (GC-MS) analysis of the leaf extracts confirmed the absence of terpene production in the transiently transformed N. benthamiana leaves. Currently we are performing a series of tests in order to troubleshoot the experiment and identify the origin of the problem. Proper functioning of the 2A peptide system implies the production of a single polycistronic mRNA, in which the coding sequences of all the genes of interest and the 2A peptide sequences form a single open reading frame (ORF). It is thus critical that no modification of the DNA sequence (nucleotide insertion/deletion or nonsense mutation) disrupts that ORF. To try to rule out the possibility that codon optimizing all the sequences and assembling them did not create any cryptic splicing sites, all the expression vector sequences that were designed, were subjected to the intron/exon pattern prediction tool GENSCAN (http://genes.mit.edu/GENSCAN.html). This analysis confirmed the absence of theoretical cryptic splicing sites in the polycistronic mRNA encoded by our expression vectors. It is as well possible that the use of the 2A peptide system is responsible for the production of non-functional proteins, as the system relies on a co-translational mechanism where ribosomes skip the synthesis of the glycyl-prolyl peptide bond at the C-terminus of a 2A peptide, leading to the cleavage between a 2A peptide and its immediate downstream peptide. As a result, the cleaved-off downstream peptide has proline at its N-terminus and the cleaved-off upstream peptide harbours the entire 2A peptide sequence minus the last proline. The additional 23 amino acids fused to the C-terminus of the terpene biosynthetic enzymes and/or the proline residue remaining on their N-terminus could thus be responsible for their loss of function. To ensure this is not the case, the constructs are being built without the 2A peptide system but with independent expression cassettes for each gene. These will be transiently expressed in N. benthamiana for functional validation. Failure of that experiment would lead to the conclusion that the Marchantia chloroplast codon optimisation could be responsible for the non-functional constructs. Introduction of rare codons into these sequences could prevent the plant from efficiently translating the recombinant mRNAs into the proteins of interest.

Follow-On Plans

We still have 1071.77£ out of the original 4000£ (as described in the table above). That money should be sufficient to cover the future cost of the Marchantia transformation as well as the cost of the GC-MS analysis of the N. benthamiana and Marchantia material that will be generated. We then do not need the additional 1000£ but we would like to keep the 1071.77£ that were not spent during the 6 first months.